Mercury ion is one of the most important metal toxins. The widespread pollution of mercury has resulted in a series of environmental and health issues. Mercury can be accumulated in the human body along the food chain and with age,that consequently leads to damaging the human central nervous system and other organs,further causes severe brain damage, cognitive problems as well as movement disorders,vision and hearing loss and even leading to death ^{[1, 2]}. Although some quantitative techniques,such as cold vapor atomic absorption spectroscopy (CVAAS) ^[3] and inductively coupled plasma mass spectroscopy (ICP-MS) ^[4],are available for detection of mercury, there has been a great need to develop sensitive,real-time and inexpensive methods for the determination of trace levels of mercury. Fluorosensors are attractive choices for this purpose due to their distinct advantages in terms of sensitivity,selectivity,and response time,and their elimination of the need of expensive instruments,highly trained personnel,and tedious maintenance ^{[5, 6, 7, 8, 9, 10]}.

Squaraines are a class of extensively studied zwitterionic dyes possessing sharp and intense absorption and fluorescence in the red and near-infrared (NIR) region ^{[11, 12, 13, 14]},and have been widely designed as NIR probes for the detection of thiol-containing amino acids ^{[15, 16]},metal ions ^{[17, 18]} and proteins ^[19]. Some analytespecific chemosensors based on squaraines that act through coordination induced charge transfer ^{[20, 21, 22]},nucleophilic attack on the electron-deficient central squaraine ring ^{[23, 24, 25]},or ion driven self-aggregation ^{[26, 27, 28, 29, 30]} have been reported. However, most of the reported cation sensors provide ‘‘turn off’’ fluorescent signals. Since monitoring a fluorescent increase from a low level is typically more reliable than monitoring a decrease from a high emission level,especially when the fluorescent changes are small, it is an important challenge to design "turn on" fluorescent probes with squaraine chromophore via new approaches.

Squaraine dyes have a high tendency to aggregate in aqueous solution,and such self-aggregation usually results in a dramatic absorption spectral broadening with fluorescence emission quenching ^[31]. The combination of the side arm of the probe with metal ions induces steric hindrance,leading to the deaggregation of the dye aggregates,companying with a fluorescence emission restoration ^[32]. Inspired by this idea,we have synthesized a new fluorescent probe USQ by introducing DTC group into a squaraine skeleton. USQ exhibits sensitive response only toward Hg²⁺ (Scheme 1).

Scheme 1

	Download: JPG larger image
Scheme 1.Rational design of the squaraine based fluorescent probe for Hg2+ via coordination induced deaggregation signaling.

Unless stated otherwise,all reagents were acquired from commercial sources and used without further purification. All solvents were purified and redistilled according to standard methods prior to use. Melting points were determined with a SGW X-4 instrument without correction. FTIR were recorded on a Perking Elmer Spectrum 2000 Fourier Transform Infrared Spectrophotometer. The ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were measured on a Bruker AV-400 spectrometer (TMS as internal standard). Electrospray ionization mass spectra (ESI-MS) were performed on a DECAX-30000 LCQ Deca XP ion trap mass spectrometer. High resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on an Agilent 6520 Accurate- Mass Q-TOF LC/MS. Fluorescent emission spectra were collected using a Cary Eclipse fluorescence spectrophotometer. Absorption spectra were measured on a Perkin Elmer Lambda 750 UV-vis spectrophotometer.

Synthesis of 3: N-Methyl-N-(2-chloroethyl)aniline (0.61 g, 3.6 mmol) and sodium diethyl dithiocarbamate (2) (0.90 g, 5.3 mmol) were dissolved in ethanol (50 mL),then a catalytic amount of potassium iodide was added. The stirred mixture was heated to reflux and the reaction was monitored by TLC. After the reaction mixture was filtered and the solvent was removed,the residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate,10:1,v/v) to give 3 as yellow oil (0.85 g),yield 86%. FTIR (KBr,cm^-1): n_max 2975,2931,2871,1599, 1505,1487,1416,1351,1269,1204,1141,985,915,748,692; ¹H NMR (400 MHz,CDCl₃): δ 1.28 (t,6H,J = 7.0 Hz),3.02 (s,3H), 3.46-3.49 (m,2H),3.60-3.64 (m,2H),3.73 (q,2H,J = 7.1 Hz),4.04 (q,2H,J = 6.9 Hz),6.69 (t,1H,J = 7.2 Hz),6.79 (δ,2H,J = 8.0 Hz), 7.21-7.25 (m,2H); ¹³C NMR (100 MHz,CDCl₃): δ 11.60,12.48, 32.96,38.12,46.78,49.61,51.66,112.14,116.37,129.21,148.79, 195.03; ESI-MS: m/z 283.4 ([M+H]⁺).

Synthesis of USQ: A mixture of semisquaric acid 4 (30 mg, 0.10 mmol) and 3 (28 mg,0.10 mmol) were dissolved in 30 mL benzene/n-BuOH (1:1,v/v) in a 100 mL round bottom flask equipped with a Dean-Stark trap. The solution was refluxed under the protection of nitrogen for 12 h. After cooling,most of the solvent was first removed under reduced pressure,and then the blue crude product was purified by column chromatography over silica gel. The elution of the column with a mixture of methylene dichloride and ethyl acetate (40:1,v/v) was used to afford the desired green squaraine dye USQ (15 mg),yield 27%. Melting point: 196-200 ℃. FTIR (KBr,cm^-1): n_max 2957,2930,1608,1585,1396, 1384,1332,1172,843,780; ¹H NMR (400 MHz,CDCl₃): d 0.99 (t, 6H,J = 7.2 Hz),1.25-1.32 (m,6H),1.36-1.45 (m,4H),1.61-1.69 (m, 4H),3.24 (s,3H),3.44 (t,4H,J = 8.0 Hz),3.51-3.56 (m,2H),3.72 (q, 2H,J = 7.2 Hz),3.79-3.83 (m,2H),4.05 (q,2H,J = 7.2 Hz),6.74 (d, 2H,J = 9.2 Hz),6.87 (d,2H,J = 9.2 Hz),8.39 (dd,4H,J = 1.6,9.2 Hz); ¹³C NMR (100 MHz,CDCl₃): d 11.58,12.50,13.86,20.24,29.62, 32.93,38.61,46.92,49.84,51.28,112.40,112.43,119.56,120.33, 132.94,133.65,153.84,153.95,183.41,187.66,189.26,194.07; ESI-MS: m/z 566.4 ([M+H]⁺); HR-ESI-MS: Calcd. for C₃₂H₄₄N₃O₂S₂ ([M+H]⁺): 566.2875,Found: 566.2898. 3. Results and discussion

USQ was prepared through a two-step process as shown in Scheme 2. N-Methyl-N-(2-chloroethyl)aniline (1) and semisquaric acid 4 were obtained according to the literature ^{[33, 34]}. The aniline derivative 3 was obtained by the treatment of sodium diethyl dithiocarbamate (2) with 1. Then a condensation reaction between compound 4 and 3 afforded the target product USQ. The new compounds were fully characterized by FTIR,¹H NMR,¹³C NMR and MS (Figs. S1-S10 in Supporting information).

Scheme 2

	Download: JPG larger image
Scheme 2.Synthesis of USQ sensor.

Squaraine dyes exhibit a high tendency to form aggregates in aqueous medium,which can significantly alter photophysical properties including a dramatic absorption change and fluorescence quenching. As shown in Fig. 1,USQ possess a strong absorption band around 640 nm in the aqueous EtOH solution with the water concentration from 0% to 70%. As the percentage of water increased to 80%,the monomer peak at around 640 nm almost disappeared and a broad band belonging to aggregates emerged. Moreover,a narrow distribution of aggregates with average size of about 104 nm was found in the aqueous solution of USQ by dynamic light scattering (DLS) techniques (Fig. S11 in Supporting information). Studies revealed that the aggregate of USQ was unstable and could be deaggregated by Hg²⁺,leading to the release of fluorescent signal. The fluorescent response of USQ toward Hg²⁺ in varied ratios of ethanol aqueous solutions has been investigated. Based on the experimental results (Fig. 2),a "turn on" fluorescent probe was designed for the detection of Hg²⁺,and EtOH-H₂O (20:80,v/v) was selected as the optimal analytical condition.

Fig. 1

	Download: JPG larger image
Fig. 1.Changes in absorbance of USQ (4.0 mmol/L) with increase of H2O percentage (0-90%) in EtOH-H2O solutions.

Fig. 2

	Download: JPG larger image
Fig. 2.Changes in fluorescence intensity of USQ (4.0 mmol/L) at 670 nm with increasing addition of Hg2+ (0-3 equiv.) in EtOH-H2O (10:90,15:85,20:80,25:75, v/v) solutions,respectively. (λex = 630 nm,slit: 5 nm/5 nm,PMT volts: 680).

The recognition behavior between USQ and Hg²⁺ was studied by absorption spectral titration (Fig. 3). Upon addition of Hg²⁺,the aggregate bands in the region of 575-700 nm were gradually reduced,and concomitant with increasing monomer absorption at 650 nm. Then,a stepwise addition of Hg²⁺ to USQ was observed through fluorescence experiments (Fig. 4). With increasing concentration of Hg²⁺,the intensity of fluorescence emission was enhanced by approximately 4-fold at 667 nm,and it presented a good linear plot (R² = 0.996,k = 2.7 × 10⁷ a.u./M) in the range of 1.0-5.0 mmol/L (0.25-1.25 equiv.) with the detection limit of 2.19 × 10^-8 mol/L (3σ/k) (Fig. S12 in Supporting information). The photostability testing of USQ has been carried out and the results showed that no photobleaching of USQ occurred under irradiation of tungsten lamp (500 W) for 8 h (Fig. S13 in Supporting information). Further kinetic studies showed that the fluorescence intensity of USQ immediately reached a maximum upon addition of Hg²⁺,indicating a fast fluorescent response of USQ toward Hg²⁺, and the optical signal kept a good stability within 30 min (Fig. S14 in Supporting information).

Fig. 3

	Download: JPG larger image
Fig. 3.The absorption spectra of USQ (4.0 μmol/L) upon addition of Hg2+ (0- 1.75 equiv.) in EtOH-H2O (20:80,v/v) solution.

Fig. 4

	Download: JPG larger image
Fig. 4.Changes in fluorescence intensity of USQ (4.0 mmol/L) with increasing addition of Hg2+ (0-3 equiv.) in EtOH-H2O (20:80,v/v) solution. Inset: The binding isotherm of USQ at 670 nm on the addition of Hg2+. (λex = 630 nm,slit: 5 nm/5 nm, PMT volts: 680).

Fig. 5 illustrates the fluorescence intensity of USQ in the presence of 50 mmol/L other interfering cations such as Li+,Na+,K+, Mg²⁺,Ca²⁺,Ba²⁺,Fe³⁺,Co²⁺,Ni²⁺,Cu²⁺,Mn²⁺,Zn²⁺,Cd²⁺,Pb²⁺,Al³⁺ and Ag⁺,and following with the addition of 4.0 mmol/L Hg²⁺ to these systems. Compared to blank solution,the changes in fluorescence intensity were not obvious in the presence of the respective cations. Instead,upon the addition of Hg²⁺,the fluorescence intensity was enhanced dramatically. These results demonstrate that the presence of above cations did not interfere with the determination of Hg²⁺,proving that USQ probe has high selectivity for Hg²⁺. In addition,the anion-dependent experiments have also been performed. As shown in Fig. 6,50 mmol/L anions including NO₃^-,ClO₄^-,SO₄ ^2-,H2PO₄^-,HPO₄ ^2-,CO₃ ^2-,HCO₃^- and CH₃COO^- did not interfere with the fluorescence enhancement toward two equiv. Hg²⁺,while only Cl^-,SCN^- and S^2- showed obvious interference due to their high affinity to Hg²⁺.

Fig. 5

	Download: JPG larger image
Fig. 5.The competition experiments of Hg2+ with other metal ions at 670 nm. Black bar: The fluorescence intensity of USQ (4.0 mmol/L) with the addition of the respective competing metal ions (50 mmol/L) in EtOH-H2O (20:80,v/v) solution. Red bar: The subsequent addition of Hg2+ (4.0 mmol/L) to the system. (λex = 630 nm, slit: 5 nm/5 nm,PMT volts: 680).

Fig. 6

	Download: JPG larger image
Fig. 6.The competition experiments of Hg2+ with different anions at 670 nm. Black bar: The fluorescence intensity of USQ (4.0 mmol/L) with the addition of the respective competing anions (50 mmol/L) in EtOH-H2O (20:80,v/v) solution. Red bar: The subsequent addition of Hg2+ (8.0 mmol/L) to the system. (λex = 630 nm, slit: 5 nm/5 nm,PMT volts: 680).

The binding mode between USQ and Hg²⁺ was observed by ¹H NMR spectrum. The ¹H NMR spectrum of USQ in CDCl₃ (Fig. 7(1)) shows eight aromatic protons at 8.39 ppm [1,8-H], 6.87 ppm [2-H] and 6.74 ppm [9-H]. Two triplet signals at 3.81 ppm [3-H] and 3.54 ppm [4-H] are assigned to N,S-methylenes, while the other two quartet signals at 4.05 ppm [6-H] and 3.72 ppm [5-H] are assigned to the methylenes of N-ethyl located on DTC. A triplet at 3.44 ppm [10-H] and a singlet at 3.24 ppm [7- H] belong to the protons of -NCH₂ and -NCH₃,respectively. After coordination with Hg²⁺,H2 and H7 signals shift to higher field (△δ = 0.05),the overlapping aromatic signals at 8.40 ppm [1,8-H] move in the opposite direction and split into a quartet,and the N,S-methylenes (3,4,5 and 6H) move to lower field incrementally. It is interesting to note that upon coordination of Hg²⁺,signals on DTC group shift obviously,while H10 and H9 belonging to N,N-dibutylanilino moiety do not change (Figs. 7(2) and (3)). These results demonstrate the coordination process between DTC group and Hg²⁺ ion along with the electronic structural change of the DTC side chain. The emission-based Job’s plot shows that a maximum emission occurred when the mole fraction of Hg²⁺ reached 0.5, which is a signature of 1:1 binding mode between USQ and Hg²⁺ (Fig. S15 in Supporting information). This is also confirmed by the appearance of a peak atm/z 829.3 assignable to [USQ-Hg²⁺-NO₃^-]⁺ (calculated for C₃₂H₄₃HgN₄O₅S₂ 829.24) in ESI mass spectrum (Fig. S16 in Supporting information). The steric hindrance of the 1:1 compλ_ex between USQ and Hg²⁺ might induce the deaggregation of the aggregates,resulting in the absorption and the fluorescence enhancement (Fig. S17 in supporting information).

Fig. 7

	Download: JPG larger image
Fig. 7.1H NMR spectra of USQ in CDCl3 with increasing concentration of (CH3COO)2Hg. The mole ratio of (CH3COO)2Hg to [USQ] is 0 (1),1 (2),2 (3),respectively.

In conclusion,based on the strategy of coordination induced deaggregation of the aggregates,a selective and sensitive fluorescence "turn on" probe for the detection of Hg²⁺ was designed and synthesized. Considering the sensing was achieved in aqueous solution,we anticipate that it will potentially serve as a fluorescent probe for Hg²⁺-related environmental and biological studies. Acknowledgments

This work was financially supported by the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (No. J1103303),the National Natural Science Foundation of China (No. 20702005),the Fujian Provincial Department of Science and Technology,China (No. 2013Y0062),Funding (Type A) from Fujian Education Department, PR China (Nos. JA12038 and JA13043) and the Science and Technology Development Fund of Fuzhou University,China (No. 600902). Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2014.04.027.